Endophilin homologous proteins involved in the regulation of energy homeostasis

ABSTRACT

The present invention discloses Endophilin homologous proteins regulating the energy homeostasis and the metabolism of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention also relates to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases and disorders, for example, but not limited to, metabolic diseases such as obesity as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea.

This invention relates to the use of nucleic sequences encodingEndophilin proteins, and the polypeptides encoded thereby, and to theuse thereof or effectors of Endophilin in the diagnosis, study,prevention, and treatment of diseases and disorders related tobody-weight regulation, for example, but not limited to, metabolicdiseases such as obesity as well as related disorders such as eatingdisorder, cachexia, diabetes mellitus, hypertension, coronary heartdisease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones,cancer, e.g. cancers of the reproductive organs, and sleep apnea.Especially preferred is the use of Endophilin nucleic acid sequences andpolypeptides or effectors of Endophilin in the modulation ofadipogenesis.

There are several metabolic diseases of human and animal metabolism,eg., obesity and severe weight loss, that relate to energy imbalancewhere caloric intake versus energy expenditure is imbalanced. Obesity isone of the most prevalent metabolic disorders in the world. It is stilla poorly understood human disease that becomes a major health problemmore and more relevant for western society. Obesity is defined as bodyweight more than 20% in excess of the ideal body weight, frequentlyresulting in a significant impairment of health. It is associated withan increased risk for cardiovascular disease, hypertension, diabetes,hyperlipidaemia and an increased mortality rate. Besides severe risks ofillness, individuals suffering from obesity are often isolated socially.

Obesity is influenced by genetic, metabolic, biochemical, psychological,and behavioral factors. As such, it is a complex disorder that must beaddressed on several fronts to achieve lasting positive clinicaloutcome. Since obesity is not to be considered as a single disorder butas a heterogeneous group of conditions with (potential) multiple causes,it is also characterized by elevated fasting plasma insulin and anexaggerated insulin response to oral glucose intake (Koltermann, J.Clin. Invest 65, 1980, 1272-1284) and a clear involvement of obesity intype 2 diabetes mellitus can be confirmed (Kopelman, Nature 404, 2000,635-643).

The molecular factors regulating food intake and body weight balance areincompletely understood. Even if several candidate genes have beendescribed which are supposed to influence the homeostatic system(s) thatregulate body mass/weight, like leptin, VCPI, VCPL, or the peroxisomeproliferator-activated receptor-gamma co-activator, the distinctmolecular mechanisms and/or molecules influencing obesity or bodyweight/body mass regulations are not known. In addition, severalsingle-gene mutations resulting in obesity have been described in mice,implicating genetic factors in the etiology of obesity (Friedman andLeibel, 1990, Cell 69: 217-220). In the ob mouse a single gene mutation(obese) results in profound obesity, which is accompanied by diabetes(Friedman et. al., 1991, Genomics 11: 1054-1062).

Therefore, the technical problem underlying the present invention was toprovide for means and methods for modulating (pathological) metabolicconditions influencing body-weight regulation and/or energy homeostaticcircuits. The solution to said technical problem is achieved byproviding the embodiments characterized in the claims.

Accordingly, the present invention relates to genes with novel functionsin body-weight regulation, energy homeostasis, metabolism, and obesity.The present invention discloses for a specific gene involved in theregulation of body-weight, energy homeostasis, metabolism, and obesity,and thus in disorders related thereto such as eating disorder, cachexia,diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer,e.g. cancers of the reproductive organs, and sleep apnea. The presentinvention describes human endophilin genes as being involved in thoseconditions mentioned above.

In this invention we particularly refer to Endophilin as humanEndophilin 3, Endophilin 1, or Endophilin 2 or mouse Endophilin 3,Endophilin 1, or Endophilin 2 or Drosophila Endophilin A or EndophilinB. or variants thereof. This includes Drosophila and mammalian,preferably human, homolog polypeptides or proteins or sequences encodingthose proteins. Polynucleotides encoding a protein with homologies toEndophilin are suitable to investigate diseases and disorders asdescribed above. Further, new compositions useful in diagnosis,treatment, and prognosis of diseases and disorders as described aboveare provided.

The endophilin (EEN; SH3p4/p8/p13) family of proteins have beendescribed to function in intracellular signalling, more specifically, inclathrin-mediated endocytosis. Studies on the endocytosis of synapticvesicles have shown the essential role of endophilin in vesicleformation (see, Ringstad et al. Neuron 1999 24(1):143-54). Endophilinsare SH3 domain-containing cytosolic proteins acting as acyltransferases.For example, Endophilin 1 is converting lysophosphatidic acid intophosphatidic acids by addition of the fatty acid arachidonate (see, forexample, review by Huttner & Schmidt Curr Opin Neurobiol 200010(5):543-51). Endophilin family members are highly expressed in brain,concentrated in nerve terminals, and found in complexes with proteinimplicated in synaptic vesicle endocytosis, the polyphosphoinositidephosphatase synaptojanin and the GTPase dynamin (see, for example,Ringstad et al. J Biol Chem 2001 2;276(44):40424-30). In addition to therole of the endophilin protein family in synaptic vesicle formation, afunction of endophilins in beta 1-adrenergic receptor signaling has beenshown (see, for example, Tang et al., Proc Natl Acad Sci USA 199926;96(22):12559-64).

So far, it has not been described that Endophilin (which particularlyrefers to human Endophilin 3, Endophilin 1, or Endophilin 2, or variantsthereof) is involved in the regulation of energy homeostasis andbody-weight regulation and related disorders, and thus, no functions inmetabolic diseases and other diseases as listed aboved have beendiscussed.

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure.

The present invention discloses that Endophilin homologous proteins areregulating the energy homeostasis and fat metabolism especially themetabolism and storage of triglycerides, and polynucleotides, whichidentify and encode the proteins disclosed in this invention. Theinvention also relates to vectors, host cells, antibodies, andrecombinant methods for producing the polypeptides and polynucleotidesof the invention. The invention also relates to the use of thesesequences in the diagnosis, study, prevention, and treatment of diseasesand disorders, for example, but not limited to, metabolic diseases suchas obesity and diabetes as well as related disorders.

The term ‘GenBank Accession number’ relates to NCBI GenBank databaseentries (Benson et al, Nucleic Acids Res. 28, 2000, 15-18).

Endophilin homologous proteins and nucleic acid molecules codingtherefore are obtainable from insect or vertebrate species, e.g. mammalsor birds. Particularly preferred are human Endophilin homologous nucleicacids, particularly nucleic acids encoding a human Endophilin 3 protein,a human Endophilin 1 protein, or a human Endophilin 2 protein. Alsopreferred are mouse Endophilin homologous nucleic acids and polypeptidesencoded thereby.

The invention particularly relates to a nucleic acid molecule encoding apolypeptide contributing to regulating the energy homeostasis and themetabolism of triglycerides, wherein said nucleic acid moleculecomprises

-   -   (a) the nucleotide sequence of human Endophilin homologous        nucleic acids, particularly nucleic acids encoding a human        Endophilin 3 protein (Genbank Accession No. NM_(—)003027;        Genbank Accession No. AF036269; EEN-B2-L1, SH3-domain GRB2-like        3); a human Endophilin 1 protein (Genbank Accession No.        NM_(—)003025; Genbank Accession No. AF036268; EEN-B1, SH3-domain        GRB2-like-1), or a human Endophilin 2 protein (Genbank Accession        No. NM 003026; Genbank Accession No. U65999; EEN, Sh3-domain        GRB2-like 2), and/or a sequence complementary thereto,    -   (b) a nucleotide sequence which hybridizes at 50° C. in a        solution containing 1×SSC and 0.1% SDS to a sequence of (a),    -   (c) a sequence corresponding to the sequences of (a) or (b)        within the degeneration of the genetic code,    -   (d) a sequence which encodes a polypeptide which is at least        85%, preferably at least 90%, more preferably at least 95%, more        preferably at least 98% and up to 99.6% identical to the amino        acid sequences of Endophilin proteins, preferably of human        Endophilin proteins (GenBank Accession Numbers NP_(—)003018,        NP_(—)003016, and NP_(—)003017),    -   (e) a sequence which differs from the nucleic acid molecule        of (a) to (d) by mutation and wherein said mutation causes an        alteration, deletion, duplication and/or premature stop in the        encoded polypeptide or    -   (f) a partial sequence of any of the nucleotide sequences of (a)        to (e) having a length of at least 15 bases, preferably at least        20 bases, more preferably at least 25 bases and most preferably        at least 50 bases.

The invention is based on the finding that Endophilin homologousproteins (herein referred to as Endophilin; preferably GenBank AccessionNumbers NP_(—)003018, NP_(—)003016, or NP_(—)003017 for the humanproteins) and the polynucleotides encoding these (for example, GenBankAccession Numbers NM_(—)003027, NM_(—)003025, or NM_(—)003026 for thehuman cDNAs), are involved in the regulation of triglyceride storage andtherefore energy homeostasis. The invention describes the use of thesecompositions comprising the nucleic acids, polypeptides and effectorsthereof, e.g. antibodies, aptamers, anti-sense molecules, ribozymes,RNAi molecules, peptides, low-molecular weight organic-molecules andother receptors recognizing the nucleic acid or the polypeptide for thediagnosis, study, prevention, or treatment of diseases and disordersrelated thereto, including metabolic diseases such as obesity as well asrelated disorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, and sleep apnea.

In this invention we demonstrate that the correct gene dose ofEndophilin is essential for maintenance of energy homeostasis. The flyDrosophila melanogaster was used as model organism for theidentification of proteins involved in the energy homeostasis.Drosophila melanogaster is one of the most intensively studied organismsin biology and serves as a model system for the investigation of manydevelopmental and cellular processes common to higher eukaryotes,including humans (see, for example, Adams et al., Science 287: 2185-2195(2000)). The success of Drosophila melanogaster as a model organism islargely due to the power of forward genetic screens to identify thegenes that are involved in a biological process (see, Johnston Nat RevGenet 3: 176-188 (2002); Rorth, Proc Natl Acad Sci USA 93: 12418-12422(1996)).

One resource for screening was a publicly available as well as aproprietary Drosophila melanogaster stock collection of EP-lines. TheP-vector of this collection has Gal4-UAS-binding sites fused to a basalpromoter that can transcribe adjacent genomic Drosophila sequences uponbinding of Gal4 to UAS-sites. This enables the EP-line collection foroverexpression of endogenous flanking gene sequences. In addition,without activation of the UAS-sites, integration of the EP-element intothe gene is likely to cause a reduction of gene activity, and allowsdetermining its function by evaluating the loss-of-function phenotype.

Triglycerides are the most efficient storage for energy in cells. Obesepeople mainly show a significant increase in the content oftriglycerides. In order to isolate genes with a function in energyhomeostasis, several thousand EP-lines were tested for theirtriglyceride content after a prolonged feeding period (see Examples formore detail). Lines with significantly changed triglyceride content wereselected as positive candidates for further analysis.

Male flies heterozygous for the integration of vectors for Drosophilalines EP(3)0593 integration were analyzed in an assay measuring thetriglyceride contents of these flies, illustrated in more detail in theEXAMPLES. The result of the triglyceride content analysis is shown inFIG. 1. The average triglyceride level of the proprietary fly collectionin which the EP(3)0593 lines was found is shown as 100% in FIG. 1 (Firstcolumn, EP-control males). The average increase of triglyceride contentof the heterozygous lethal Drosophila line EP(3)0593(referred to as‘EP(3)0593’ in this invention) is 100% (see FIG. 1, second column,‘EP(3)0593/TM3,Sb males’). It was found in this invention thatheterozygous EP(3)0593 flies have a significant higher triglyceridecontent than the control flies tested. The increase of triglyceridecontent due to the potential loss of a gene function suggests potentialgene activities in energy homeostasis in a dose dependent manner thatcontrols the amount of energy stored as triglycerides.

Nucleic acids encoding the Endophilin protein of the present inventionwere identified using a plasmid-rescue technique. Genomic DNA sequenceswere isolated that are localised to the EP vector (herein EP(3)0593)integration. Using those isolated genomic sequences public databaseslike Berkeley Drosophila Genome Project (GadFly) were screened therebyconfirming the heterozygous lethal integration side of the EP(3)0593vector 45 base pairs 5′ of the cDNA of the Endophilin A gene, identifiedas Berkeley Drosophila Genome Project Accession Nr. CG1 4296 (FIG. 2).FIG. 2 shows the molecular organisation of this gene locus. Thechromosomal localization site of the integration of the vector ofEP(3)0593 is at gene locus 3R, 91 D4. In FIG. 2, genomic DNA sequence isrepresented as a black dotted line in the middle that includes theintegration site of EP(3)0593. Numbers represent the coordinates of thegenomic DNA (starting at position 14653366 on chromosome 3R, ending atposition 14678366 on chromosome 3R). Grey bars on the two “cDNA”-linesrepresent the predicted genes (as predicted by the Berkeley DrosophilaGenome Project, GadFly and by Magpie). Predicted exons of the DrosophilacDNA (Berkeley Drosophila Genome Project Accession Nr. CG14296) areshown as dark grey bars and predicted introns as light grey bars. Theboxes referring to expressed sequence tag (EST) clones ‘DGC GH12907’(dark grey bars) and to EST clones ‘Clot 5557_(—)3 & 1’ (light greybars) show that endophilin is a transcribed gene.

Expression profiling studies (see Examples for more detail) confirm theparticular relevance of Endophilin 3 and Endophilin 1 as regulators ofenergy metabolism in mammals. In comparison to Endophilin 2, which israther ubiquitously expressed, both Endophilin 3 and Endophilin 1transcripts are more restricted in neuronal tissues of mammals. However,both Endophilin 3 and Endophilin 1 are also clearly expressed in whiteadipose tissue (WAT) and brown adipose tissue (BAT) (see FIG. 4A andFIG. 5A, respectively), indicating a role in the regulation of energyhomeostasis.

Further, we show that mammalian homologues of the Drosophila Endophilingene, particularly Endophilin 3 and. Endophilin 1, are regulated bymetabolic conditions such as fasting and genetically induced obesity.For example, the expression of Endophilin 3 is strongly upregulated inliver of fasted mice (see FIG. 4B). In addition, a marked downregulationcan be observed in the metabolically active tissue (for example, brownadipose tissue (BAT)) of genetically obese (ob/ob) as well as of fastedmice (see FIG. 4B). Endophilin 1 is strongly downregulated in whiteadipose tissue (WAT) of genetically obese db/db mice (see FIG. 5B).

The most prominent response with regard to metabolically active tissuescan be seen in mice kept under a high fat diet. In those mice, theexpression of Endophilin 3 as well as Endophilin 1 is significantlyreduced in white adipose tissue (see FIG. 4C and FIG. 5C, respectively),supporting the finding that Endophilin 3 and/or Endophilin 1 is amodulator (for example, inhibitor) of adipogenesis.

In addition, we show in this invention that the Endophilin 3 mRNA isstrongly down-regulated during adipocyte differentiation in vitro (seeEXAMPLES for more detail), demonstrating a role as modulator (forexample, inhibitor) of adipocyte lipid accumulation. With regard tochanges in expression intensity during the differentiation ofpreadipocytes to adipocytes, a strong reduction in relative signalintensity can be observed for Endophilin 3 during the in vitrodifferentiation program of 3T3-L1 as well as 3T3-F442A cells (see FIG.4D and FIG. 4E).

Thus, we conclude that Endophilin 3 (or variants thereof) or Endophilin1 (or variants thereof) has a function in the metabolism of matureadipocytes.

The present invention is further describing a polypeptide comprising theamino acid sequence of Endophilin. A comparison (Clustal X 1.8 analysis)between the Endophilin proteins of different species (human andDrosophila) was conducted (See FIG. 3). The sequence similaritiesbetween Drosophila Endophilin A (GadFly Accession Number CG14296) andhuman homologs (for Endophilin 3, NP_(—)003018; for Endophilin 1,NP_(—)003016; for Endophilin 2, NP_(—)003017) are as described below:

-   -   CG14296 vs. GenBank Accession No. NP_(—)003018 50% identity, 66%        similarity (366 amino acids)    -   CG14296 vs. GenBank Accession No. NP_(—)003016 49% identity, 67%        similarity (370 amino acids)    -   CG14296 vs. GenBank Accession No. NP_(—)003017 48% identity, 65%        similarity (369 amino acids)

Based upon homology, an Endophilin protein of the invention and eachhomologous protein or peptide may share at least some activity.

No functional data for such proteins relating to the regulation of bodyweight control and related metabolic diseases such as obesity areavailable in the prior art. Our Drosophila data as well as results fromthe expression profiling in different mouse models clearly show thatEndophilin is involved in the regulation of the metabolism in fly andmammals. In addition, as shown in FIG. 7C, the basal glucose uptake ofcells over-expressing endophillin 3 is significantly increased duringadipogenesis. This increase in glucose and therefore energy uptake ofthe cells is most likely the reason for the increased glycogen andtriglyceride levels during adipocyte differentiation (see FIGS. 7A and7B, respectively). Endophillin 3 does not seem to influence insulinstimulated glucose uptake but clearly has an effect on the glucoseuptake of adipocytes, confirming a role in diabetes and relatedmetabolic disorders.

The invention also encompasses polynucleotides that encode Endophilinand homologous proteins. Accordingly, any nucleic acid sequence, whichencodes the amino acid sequences of Endophilin, can be used to generaterecombinant molecules that express Endophilin. In a particularembodiment, the invention encompasses human Endophilin homologousnucleic acids, particularly nucleic acids encoding a human Endophilin 3protein, a human Endophilin 1 protein, or a human Endophilin 2 protein.Also preferred are mouse Endophilin homologous nucleic acids andpolypeptides encoded thereby. It will be appreciated by those skilled inthe art that as a result of the degeneracy of the genetic code, amultitude of nucleotide sequences encoding Endophilin, some bearingminimal homology to the nucleotide sequences of any known and naturallyoccurring gene, may be produced. Thus, the invention contemplates eachand every possible variation of nucleotide sequence that could be madeby selecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequences of naturally occurringEndophilin, and all such variations are to be considered as beingspecifically disclosed. Although nucleotide sequences which encodeEndophilin and its variants are preferably capable of hybridising to thenucleotide sequences of the naturally occurring Endophilin underappropriately selected conditions of stringency, it may be advantageousto produce nucleotide sequences encoding Endophilin or its derivativespossessing a substantially different codon usage. Codons may be selectedto increase the rate at which expression of the peptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilised by the host. Otherreasons for substantially altering the nucleotide sequence encodingEndophilin and its derivatives without altering the encoded amino acidsequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequences. The invention alsoencompasses production of DNA sequences, or portions thereof, whichencode Endophilin and its derivatives, entirely by synthetic chemistry.After production, the synthetic sequence may be inserted into any of themany available expression vectors and cell systems using reagents thatare well known in the art at the time of the filing of this application.Moreover, synthetic chemistry may be used to introduce mutations into asequence encoding Endophilin any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridising to the claimed nucleotide sequences, and inparticular, human Endophilin homologous nucleic acids, particularlynucleic acids encoding a human Endophilin 3 protein, a human Endophilin1 protein, or a human Endophilin 2 protein under various conditions ofstringency. Hybridisation conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987: Methods Enzymol. 152:399-407) andKimmel, A. R. (1987, Methods Enzymol. 152:507-511), and may be used at adefined stringency. Preferably, hybridization under stringent conditionsmeans that after washing for 1 h with 1×SSC and 0.1% SDS at 50° C.,preferably at 55° C., more preferably at 62° C. and most preferably at68° C., particularly for 1 h in 0.2×SSC and 0.1% SDS at 50° C.,preferably at 55° C., more preferably at 62° C. and most preferably at68° C., a positive hybridization signal is observed. Altered nucleicacid sequences encoding Endophilin which are encompassed by theinvention include deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent Endophilin.

The encoded proteins may also contain deletions, insertions, orsubstitutions of amino acid residues, which produce a silent change andresult in a functionally equivalent Endophilin. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological activity ofEndophilin is retained. For example, negatively charged amino acids mayinclude aspartic acid and glutamic acid; positively charged amino acidsmay include lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; phenylalanine and tyrosine. Furthermore, theinvention relates to peptide fragments of the proteins or derivativesthereof such as cyclic peptides, retro-inverso peptides or peptidemimetics having a length of at least 4, preferably 6 and up to 50 aminoacids.

Also included within the scope of the present invention are alleles ofthe genes encoding Endophilin. As used herein, an “allele” or “allelicsequence” is an alternative form of the gene, which may result from atleast one mutation in the nucleic acid sequence. Alleles may result inaltered mRNAs or polypeptides whose structures or function may or maynot be altered. Any given gene may have none, one, or many allelicforms. Common mutational changes, which give rise to alleles, aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymerase1, SEQUENASE DNA Polymerase (US Biochemical Corp, Cleveland Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproof-reading exonucleases such as the ELONGASE Amplification System(GIBCO/BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno Nev.),Peltier thermal cycler (PTC200; MJ Research, Watertown, Mass.) and theABI 377 DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding Endophilin may be extended utilisinga partial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). Inverse PCR may also be used to amplify or extendsequences using divergent primers based on a known region (Triglia, T.et al. (1988) Nucleic Acids Res. 16:8186). Another method which may beused is capture PCR which involves PCR amplification of DNA fragmentsadjacent to a known sequence in human and yeast artificial chromosomeDNA (Lagerstrom, M. et al. (PCR Methods Applic. 1:111-119). Anothermethod which may be used to retrieve unknown sequences is that ofParker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences, which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions. Capillary electrophoresis systems,which are commercially available, may be used to analyse the size orconfirm the nucleotide sequence of sequencing or PCR products. Inparticular, capillary sequencing may employ flowable polymers forelectrophoretic separation, four different fluorescent dyes (one foreach nucleotide) which are laser activated, and detection of the emittedwavelengths by a charge coupled devise camera. Output/light intensitymay be converted to electrical signal using appropriate software (e.g.GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer) and the entire processfrom loading of samples to computer analysis and electronic data displaymay be computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA, which might bepresent in limited amounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode Endophilin, or fusion proteins orfunctional equivalents thereof, may be used in recombinant DNA moleculesto direct expression of Endophilin in appropriate host cells. Due to theinherent degeneracy of the genetic code, other DNA sequences, whichencode substantially the same, or a functionally equivalent amino acidsequence may be produced and these sequences may be used to clone andexpress Endophilin. As will be understood by those of skill in the art,it may be advantageous to produce Endophilin-encoding nucleotidesequences possessing non-naturally occurring codons. For example, codonspreferred by a particular prokaryotic or eukaryotic host can be selectedto increase the rate of protein expression or to produce a recombinantRNA transcript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence. The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterEndophilin encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding Endophilin may be ligated toa heterologous sequence to encode a fusion protein. For example, toscreen peptide libraries for inhibitors of Endophilin activities, it maybe useful to produce chimeric Endophilin proteins that can be recognisedby a commercially available antibodies. A fusion protein may also beengineered to contain a cleavage site located between the Endophilinencoding sequence and the heterologous protein sequences, so thatEndophilin may be cleaved and purified away from the heterologousmoiety. In another embodiment, sequences encoding Endophilin may besynthesised, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.7:215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.7:225-232). Alternatively, the proteins themselves may be produced usingchemical methods to synthesise the amino acid sequence of Endophilin, ora portion thereof. For example, peptide synthesis can be performed usingvarious solid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A peptide synthesiser (Perkin Elmer). The newly synthesisedpeptide may be substantially purified by preparative high performanceliquid chromatography (e.g., Creighton, T. (1983) Proteins, Structuresand Molecular Principles, W H Freeman and Co., New York, N.Y.). Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure;Creighton, supra). Additionally, the amino acid sequences of Endophilin,or any part thereof, may be altered during direct synthesis and/orcombined using chemical methods with sequences from other proteins, orany part thereof, to produce a variant polypeptide.

In order to express a biologically active Endophilin, the nucleotidesequences encoding Endophilin functional equivalents, may be insertedinto appropriate expression vectors, i.e., a vector, which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods, which are well known to those skilled in theart, may be used to construct expression vectors containing sequencesencoding Endophilin and appropriate transcriptional and translationalcontrol elements. These methods include in vitro recombinant DNAtechniques synthetic techniques, and in vivo genetic recombination. Suchtechniques are described in Sambrook, J. et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,and Ausubel, F. M. et al. (1989) Current Protocols in. MolecularBiology, John Wiley & Sons, New York, N.Y.

A variety of expression vector/host systems may be utilised to containand express sequences encoding Endophilin. These include, but are notlimited to, micro-organisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or PBR322 plasmids); or animal cellsystems. The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector-enhancers, promoters, 5′ and 3′untranslated regions which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilised, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters and enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters and leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequences encodingEndophilin, vectors based on SV40 or EBV may be used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for Endophilin. For example, when largequantities of Endophilin are needed for the induction of antibodies,vectors, which direct high level expression of fusion proteins that arereadily purified, may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asthe BLUESCRIPT phagemid (Stratagene), in which the sequence encodingEndophilin may be ligated into the vector in frame with sequences forthe amino-terminal Met and the subsequent 7 residues of β-galactosidaseso that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. Vectorsof the pGEX series (Amersham Bioscience, Uppsala, Sweden) may also beused to express foreign polypeptides as fusion proteins with GlutathioneS-Transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. In another expression system, the coding sequence ofendophilin may be expressed in frame with six or more consequtivehistidin residues, which may be located close to the amino terminus,carboxy terminal or internally. A variety of vectors, which include, butare not limited to, the pQE series (Qiagen, Hilden, Germany), pBAD andpTrc series (Invitrogen, Carlsbad, Calif.) and pET series (Novagen,Madison, Wis.) may be suitable for expression of endophilin in E. coliand a subsequent purification from the lysed cells by imobilized metalion affinity chromatography (IMAC).

Proteins made in such systems may be designed to include heparin,thrombin, or factor XA protease cleavage sites so that the clonedpolypeptide of interest can be released from the GST moiety at will. Inthe yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al., (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding Endophilin may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express Endophilin. For example, inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encodingEndophilin may be cloned into a non-essential region of the virus, suchas the polyhedrin gene, and place under control of the polyhedrinpromoter. Successful insertions of Endophilin will render the polyhedringene inactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells of Trichoplusia larvae in which Endophilin may beexpressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.91:3224-3227).

In mammalian host cells, a number of non-viral-based or viral-basedexpression systems may be utilised. In cases where an adenovirus is usedas an expression vector, sequences encoding Endophilin may be ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anon-essential El or E3 region of the viral genome may be used to obtainviable viruses which are capable of expressing Endophilin in infectedhost cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci.81:3655-3659). In addition, transcription enhancers, such as the Roussarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding Endophilin. Such signals include theATG initiation codon and adjacent sequences. In cases where sequencesencoding Endophilin, its initiation codons, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressEndophilin may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells, whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type. Any number of selection systemsmay be used to recover transformed cell lines. These include, but arenot limited to, the herpes simplex virus thymidine kinase (Wigler, M. etal. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy,I. et al. (1980) Cell 22:81 7-23) genes, which can be employed in tk-oraprt-,cells, respectively. Also, antimetabolite, antibiotic or herbicideresistance can be used as the basis for selection; for example, dhfrwhich confers resistance to methotrexate (Wigler, M. et al. (1980) Proc.Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to theaminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J.Mol. Biol. 150:1-14) and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively(Murry, supra). Additional selectable genes have been described, forexample, trpB, which allows cells to utilise indole in place oftryptophan, or hisD, which allows cells to utilise histinol in place ofhistidine (Hartman, S. C. and R. C. Mulligan (1 988) Proc. Natl. Acad.Sci. 85:8047-51). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, β-glucuronidase and itssubstrate GUS, and luciferase and its substrate luciferin, being widelyused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol.55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequences encoding Endophilinare inserted within a marker gene sequence, recombinant cells containingsequences encoding Endophilin can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem withsequences encoding Endophilin under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well. Alternatively,host cells, which contain the nucleic acid sequences encoding Endophilinand express Endophilin, may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA, or DNA-RNA hybridisation and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

The presence of polynucleotide sequences encoding Endophilin can bedetected by DNA-DNA or DNA-RNA hybridisation and/or amplification usingprobes or portions or fragments of polynucleotides encoding Endophilin.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding Endophilinto detect transformants containing DNA or RNA encoding Endophilin. Asused herein “oligonucleotides” or “oligomers” refer to a nucleic acidsequence of at least about 10 nucleotides and as many as about 60nucleotides, preferably about 15 to 30 nucleotides, and more preferablyabout 20-25 nucleotides, which can be used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofEndophilin, using either polyclonal or monoclonal antibodies specificfor the protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilising monoclonal antibodies reactive to two non-interfering epitopeson Endophilin is preferred, but a competitive binding assay may beemployed. These and other assays are described, among other places, inHampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APSPress, St Paul, Minn.) and Maddox, D. E. et al. (1983;, J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labelled hybridisation or PCR probesfor detecting sequences related to polynucleotides encoding Endophilininclude oligo-labelling, nick translation, end-labelling or PCRamplification using a labelled nucleotide.

Alternatively, the sequences encoding Endophilin, or any portionsthereof may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesise RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3, or SP6 and labelled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.);and U.S. Biochemical Corp., (Cleveland, Ohio).

Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, co-factors, inhibitors, magneticparticles, and the like.

Host cells transformed with nucleotide sequences encoding Endophilin maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeEndophilin may be designed to contain signal sequences, which directsecretion of Endophilin through a prokaryotic or eukaryotic cellmembrane. Other recombinant constructions may be used to join sequencesencoding Endophilin to nucleotide sequence encoding a polypeptidedomain, which will facilitate purification of soluble proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilised metals, protein A domains that allowpurification on immobilised immunoglobulin, and the domain utilised inthe FLAG extension/affinity purification system (Immunex Corp., Seattle,Wash.) The inclusion of cleavable linker sequences such as thosespecific for Factor XA or Enterokinase (Invitrogen, San Diego, Calif.)between the purification domain and Endophilin may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing Endophilin and a nucleic acid encoding 6histidine residues preceding a Thioredoxine or an Enterokinase cleavagesite. The histidine residues facilitate purification on IMIAC(immobilised metal ion affinity chromatography as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3: 263-281)) while the Enterokinasecleavage site provides a means for purifying Endophilin from the fusionprotein. A discussion of vectors which contain fusion proteins isprovided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453). Inaddition to recombinant production, fragments of Endophilin may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431Apeptide synthesiser (Perkin Elmer). Various fragments of Endophilin maybe chemically synthesised separately and combined using chemical methodsto produce the full length molecule.

Diagnostics and Therapeutics

The data disclosed in this invention show that the nucleic acids andproteins of the invention and effector molecules thereof are useful indiagnostic and therapeutic applications implicated, for example but notlimited to, in metabolic disorders such as obesity as well as relateddisorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, and sleep apnea. Hence, diagnostic and therapeuticuses for the Endophilin nucleic acids and proteins of the invention are,for example but not limited to, the following: (i) protein therapeutic,(ii) small molecule drug target, (iii) antibodytarget (therapeutic,diagnostic, drug targeting/cytotoxic antibody), (iv) diagnostic and/orprognostic marker,. (v) gene therapy (gene delivery/gene ablation), (vi)research tools, and (vii) tissue regeneration in vitro and in vivo(regeneration for all these tissues and cell types composing thesetissues and cell types derived from these tissues). A particularlypreferred application is the modulation, e.g. inhibition ofadipogenesis.

The nucleic acids and proteins of the invention and effectors thereofare useful in diagnostic and therapeutic applications implicated invarious applications as described below. For example, but not limitedto, cDNAs encoding the Endophilin proteins of the invention andparticularly their human homologues may be useful in gene therapy, andthe Endophilin proteins of the invention and particularly their humanhomologues may be useful when administered to a subject in need thereof.By way of non-limiting example, the compositions of the presentinvention will have efficacy for treatment of patients suffering from,for example, but not limited to, in metabolic disorders as describedabove.

The nucleic acid(s) encoding the Endophilin protein(s) of the invention,or fragments thereof, may further be useful in diagnostic applications,wherein the presence or amount of the nucleic acids or the proteins areto be assessed. These materials are further useful in the generation ofantibodies that bind immunospecifically to the substances of theinvention for use in therapeutic or diagnostic methods.

For example, in one aspect, antibodies which are specific for Endophilinmay be used directly as an antagonist, or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissue which express Endophilin. The antibodies may be generated usingmethods that are well known in the art. Such antibodies may include, butare not limited to, polyclonal, monoclonal, chimerical, single chain,Fab fragments, and fragments produced by a Fab expression library.Neutralising antibodies, (i.e., those which inhibit dimer formation) areespecially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunised by injectionwith Endophilin any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminiumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in human, BCG (BacilleCalmette-Guerin) and Corynebacterium parvum are especially preferable.It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to Endophilin have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of Endophilin amino acids may be fused with those ofanother protein such as keyhole limpet hemocyanin and antibody producedagainst the chimeric molecule.

Monoclonal antibodies to Endophilin may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Köhler, G. et al. (1975) Nature 256:495-497;Kozbor,. D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol.Cell Biol. 62:109-120). In addition, techniques developed for theproduction of “chimeric antibodies”, the splicing of mouse antibodygenes to human antibody genes to obtain a molecule with appropriateantigen specificity and biological activity can be used (Morrison, S. L.et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. etal. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature314:452-454). Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce Endophilin- and -specific single chain antibodies. Antibodieswith related specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries (Burton, D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments, which contain specific binding sites for Endophilin,may also be generated. For example, such fragments include, but are notlimited to, the F(ab′)₂ fragments which can be produced by Pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding and immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between Endophilin and its specific antibody. A two-site,monoclonal-based immunoassay utilising monoclonal antibodies reactive totwo non-interfering Endophilin epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingEndophilin, or any fragment thereof, or nucleic acid effector molecules,such as aptamers, anti-sense molecules, ribozymes or RNAi molecules maybe used for therapeutic purposes. In one aspect, aptamers, i.e. nucleicacid molecules, which are capable of binding to an Endophilin proteinand modulating its activity, may be generated by a screeningand-selection procedure involving the use of combinatorial nucleic acidlibraries.

In a further aspect, antisense molecules to the polynucleotide encodingEndophilin may be used in situations in which it would be desirable toblock the transcription of the mRNA. In particular, cells may betransformed with sequences complementary to polynucleotides encodingEndophilin. Thus, antisense molecules may be used to modulate Endophilinactivity, or to achieve regulation of gene function. Such technology isnow well know in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding Endophilin. Expression vectorsderived from retroviruses, adenovirus, herpes or vaccinia viruses, orfrom various bacterial plasmids may be used for delivery of nucleotidesequences to the targeted organ, tissue or cell population. Methods,which are well known to those skilled in the art, can be used toconstruct recombinant vectors, which will express antisense moleculescomplementary to the polynucleotides of the gene encoding Endophilin.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra). Genes encoding Endophilin can be turned off bytransforming a cell or tissue with expression vectors which express highlevels of polynucleotide or fragment thereof which encodes Endophilin.Such constructs may be used to introduce untranslatable sense orantisense sequences into a cell. Even in the absence of integration intothe DNA, such vectors may continue to transcribe RNA molecules untilthey are disabled by endogenous nucleases. Transient expression may lastfor a month or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, e.g. DNA, RNA, or nucleic acid analoguessuch as PNA, to the control regions of the gene encoding Endophilin,i.e., the promoters, enhancers, and introns. Oligonucleotides derivedfrom the transcription initiation site, e.g., between positions −10 and+10 from the start site, are preferred. Similarly, inhibition can beachieved using “triple helix” base-pairing methodology. Triple helixpairing is useful because it cause inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In; Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Theantisense molecules may also be designed to block translation of mRNA bypreventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyse thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridisation of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage.Examples, which may be used, include engineered hammerhead motifribozyme molecules that can be specifically and efficiently catalyseendonucleolytic cleavage of sequences encoding Endophilin. Specificribozyme cleavage sites within any potential RNA target are initiallyidentified by scanning the target molecule for ribozyme cleavage siteswhich include the following sequences: GUA, GUU, and GUC. Onceidentified, short RNA sequences of between 15 and 20 ribonucleotidescorresponding to the region of the target gene containing the cleavagesite may be evaluated for secondary structural features which may renderthe oligonucleotide inoperable. The suitability of candidate targets mayalso be evaluated by testing accessibility to hybridisation withcomplementary oligonucleotides using ribonuclease protection assays.

Nucleic acid effector molecules, e.g. antisense molecules and ribozymesof the invention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesising oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding Endophilin. Such DNA sequences may be incorporated into avariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesise antisense RNAconstitutively or inducibly can be introduced into cell lines, cells, ortissues. RNA molecules may be modified to increase intracellularstability and half-life. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ endsof the molecule or the use of phosphorothioate or 2′ O-methyl ratherthan phosphodiesterase linkages within the backbone of the molecule.This concept is inherent in the production of PNAs and can be extendedin all of these molecules by the inclusion of non-traditional bases suchas inosine, queosine, and wybutosine, as well as acetyl-, methyl-,thio-, and similarly modified forms of adenine, cytidine, guanine,thymine, and uridine which are not as easily recognised by endogenousendonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods, which are well known in the art. Any of thetherapeutic methods described above may be applied to any suitablesubject including, for example, mammals such as dogs, cats, cows,horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of Endophilin, antibodiesto Endophilin, mimetics, agonists, antagonists, or inhibitors ofEndophilin. The compositions may be administered alone or in combinationwith at least one other agent, such as stabilising compound, which maybe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones. The pharmaceuticalcompositions utilised in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations which, can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.). Pharmaceutical compositions can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilising processes. Thepharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulphuric,acetic, lactic, tartaric, malic, succinic, etc. After pharmaceuticalcompositions have been prepared, they can be placed in an appropriatecontainer and labelled for treatment of an indicated condition. Foradministration of Endophilin, such labelling would include amount,frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compounds, the therapeutically effective does can beestimated initially either in cell culture assays, e.g., of preadipocytecell lines, or in animal models, usually mice, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. A therapeutically effective dose refers to that amount of activeingredient, for example Endophilin, fragments thereof or antibodies ofEndophilin, which is sufficient for treating a specific condition.Therapeutic efficacy and toxicity may be determined by standardpharmaceutical procedures in cell cultures or experimental animals, e.g.ED50 (the dose therapeutically effective in 50% of the population) andLD50 (the dose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50. Pharmaceutical compositions, whichexhibit large therapeutic indices, are preferred. The data obtained fromcell culture assays and animal studies is used in formulating a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage from employed, sensitivity of the patient, andthe route of administration. The exact dosage will be determined by thepractitioner, in light of factors related to the subject that requirestreatment. Dosage and administration are adjusted to provide sufficientlevels of the active moiety or to maintain the desired effect. Factors,which may be taken into account, include the severity of the diseasestate, general health of the subject, age, weight, and gender of thesubject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long-acting pharmaceutical compositions may be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation. Normaldosage amounts may vary from 0.1 to 100,000 micrograms, up to a totaldose of about 1 g, depending upon the route of administration. Guidanceas to particular dosages and methods of delivery is provided in theliterature and generally available to practitioners in the art. Thoseskilled in the art employ different formulations for nucleotides thanfor proteins or their inhibitors. Similarly, delivery of polynucleotidesor polypeptides will be specific to particular cells, conditions,locations, etc.

In another embodiment, antibodies which specifically bind Endophilin maybe used for the diagnosis of conditions or diseases characterised by orassociated with over- or underexpression of Endophilin, or in assays tomonitor patients being treated with Endophilin, agonists, antagonists orinhibitors. The antibodies useful for diagnostic purposes may beprepared in the same manner as those described above for therapeutics.Diagnostic assays for Endophilin include methods, which utilise theantibody and a label to detect Endophilin in human body fluids orextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labelled by joining them, either covalently ornon-covalently, with a reporter molecule. A wide variety of reportermolecules which are known in the art may be used several of which aredescribed above.

A variety of protocols including ELISA, RIA, and FACS for measuringEndophilin are known in the art and provide a basis for diagnosingaltered or abnormal levels of Endophilin expression. Normal or standardvalues for Endophilin expression are established by combining bodyfluids or cell extracts taken from normal mammalian subjects, preferablyhuman, with antibody to Endophilin under conditions suitable for complexformation. The amount of standard complex formation may be quantified byvarious methods, but preferably by photometric means. Quantities ofEndophilin expressed in control and disease samples e.g. from biopsiedtissues are compared with the standard values. Deviation betweenstandard and subject values establishes the parameters for diagnosingdisease.

In another embodiment of the invention, the polynucleotides encodingEndophilin may be used for diagnostic purposes. The polynucleotides,which may be used, include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofEndophilin may be correlated with disease. The diagnostic assay may beused to distinguish between absence, presence, and excess expression ofEndophilin, and to monitor regulation of Endophilin levels duringtherapeutic intervention.

In one aspect, hybridisation with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding Endophilin closely related molecules, may be used to identifynucleic acid sequences which encode Endophilin. The specificity of theprobe, whether it is made from a highly specific region, e.g., uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridisation or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding Endophilin, alleles, or related sequences. Probes mayalso be used for the detection of related sequences, and shouldpreferably contain at least 50% of the nucleotides from any of theEndophilin encoding sequences. The hybridisation probes of the subjectinvention may be DNA or RNA and are preferably derived from thenucleotide sequence of human Endophilin homologous nucleic acids,particularly nucleic acids encoding a human Endophilin 3 protein, ahuman Endophilin 1 protein, or a human Endophilin 2 protein or from agenomic sequence including promoter, enhancer elements, and introns of anaturally occurring Endophilin gene. Means for producing specifichybridisation probes for DNAs encoding Endophilin include the cloning ofnucleic acid sequences encoding Endophilin derivatives into vectors forthe production of mRNA probes. Such vectors are known in the art,commercially available, and may be used to synthesise RNA probes invitro by means of the addition of the appropriate RNA polymerases andthe appropriate labelled nucleotides. Hybridisation probes may belabelled by a variety of reporter groups, for example, radionuclidessuch as ³²P or ³⁵S, or enzymatic labels, such as alkaline phosphatasecoupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotide sequences encoding Endophilin may be used for thediagnosis of conditions or diseases, which are associated withexpression of Endophilin. Examples of such conditions or diseasesinclude, but are not limited to, pancreatic diseases and disorders,including diabetes. Polynucleotide sequences encoding Endophilin mayalso be used to monitor the progress of patients receiving treatment forpancreatic diseases and disorders, including diabetes. Thepolynucleotide sequences encoding Endophilin may be used in Southern orNorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilisingfluids or tissues from patient biopsies to detect altered Endophilinexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding Endophilin maybe useful in assays that detect activation or induction of variousmetabolic diseases such as obesity as well as related disorders such aseating disorder, cachexia, diabetes mellitus, hypertension, coronaryheart disease, hypercholesterolemia, dyslipidemia, osteoarthritis,gallstones, cancers of the reproductive organs, and sleep apnea. Thenucleotide sequences encoding Endophilin may be labelled by standardmethods, and added to a fluid or tissue sample from a patient underconditions suitable for the formation of hybridisation complexes. Aftera suitable incubation period, the sample is washed and the signal isquantitated and compared with a standard value. If the amount of signalin the biopsied or extracted sample is significantly altered from thatof a comparable have hybridised with nucleotide sequences in the sample,and the presence of altered levels of nucleotide sequences encodingEndophilin in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of Endophilin, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes Endophilin, underconditions suitable for hybridisation or amplification. Standardhybridisation may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease. Once disease is established and a treatment protocol isinitiated, hybridisation assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that, which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to metabolic diseases such as obesity as well as relateddisorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancers of the reproductiveorgans, and sleep apnea the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the pancreatic diseases and disorders.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding Endophilin may involve the use of PCR. Such oligomersmay be chemically synthesised, generated enzymatically, or produced froma recombinant source. Oligomers will preferably consist of twonucleotide sequences, one with sense orientation (5′.fwdarw.3′) andanother with antisense (3′.rarw.5′), employed under optimised conditionsfor identification of a specific gene or condition. The same twooligomers, nested sets of oligomers, or even a degenerate pool ofoligomers may be employed under less stringent conditions for detectionand/or quantification of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression ofEndophilin include radiolabelling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P. C. et al.(1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.Biochem. 212:229-236). The speed of quantification of multiple samplesmay be accelerated by running the assay in an ELISA format where theoligomer of interest is presented in various dilutions and aspectrophotometric or colorimetric response gives rapid quantification.

In another embodiment of the invention, the nucleic acid Endophilinsequences, which encode Endophilin, may also be used to generatehybridisation probes, which are useful for mapping the naturallyoccurring genomic sequence. The sequences may be mapped to a particularchromosome or to a specific region of the chromosome using well knowntechniques. Such techniques include FISH, FACS, or artificial chromosomeconstructions, such as yeast artificial chromosomes, bacterialartificial. chromosomes, bacterial P1 constructions or single chromosomecDNA libraries as reviewed in Price, C. M. (1993) Blood Rev. 7:127-134,and Trask, B. J. (1991) Trends Genet. 7:149-154. FISH (as described inVerma et al. (1988) Human Chromosomes: A Manual of Basic Techniques,Pergamon Press, New York, N.Y.) may be correlated with other physicalchromosome mapping techniques and genetic map data. Examples of geneticmap data can be found in the 1994 Genome Issue of Science (265:1981f).Correlation between the location of the gene encoding Endophilin on aphysical chromosomal map and a specific disease, or predisposition to aspecific disease, may help to delimit the region of DNA associated withthat genetic disease.

The nucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals. An analysis of polymorphisms, e.g. single nucleotidepolymorphisms may be carried out. Further, in situ hybridization ofchromosomal preparations and physical mapping techniques such as linkageanalysis using established chromosomal markers may be used for extendinggenetic maps. Often the placement of a gene on the chromosome of anothermammalian species, such as mouse, may reveal associated markers even ifthe number or arm of a particular human chromosome is not known. Newsequences can be assigned to chromosomal arms or parts thereof, byphysical mapping. This provides valuable information to investigatorssearching for disease genes using positional cloning or other genediscovery techniques. Once the disease or syndrome has been crudelylocalized by genetic linkage to a particular genomic region, forexample, AT to 11q22-23 (Gatti, R. A. et al. (1988) Nature 336:577-580),any sequences mapping to that area may represent associated orregulatory genes for further investigation. The nucleotide sequences ofthe subject invention may-also be used to detect differences in thechromosomal location due to translocation, inversion, etc. among normal,carrier or affected individuals.

In another embodiment of the invention, the proteins of the invention,its catalytic or immunogenic fragments or oligopeptides thereof, an invitro model, a genetically altered cell or animal, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. One can identify effectors, e.g. receptors, enzymes, ligandsor substrates that bind to, modulate or mimic the action of one or moreof the proteins of the invention. The protein or fragment thereofemployed in such screening may be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. Theformation of binding complexes, between the proteins of the inventionand the agent tested, may be measured. Agents can also be identified,which either directly or indirectly, influence the activity of theproteins of the invention. Target mechanisms could for example includethe lysophosphatidic acid acyl transferase (LPAAT) activity ofendophilin (see, Schmidt et al.) 1999, Nature 401: 123-124). Assaysaimed at determination of LPAAT activity are well known in the art, i.e.the transfer of arachidonate from the radioactive labeled donorArachidonoyl CoA to lysophosphatidic acid could be quantified after thinlayer chromatography of the lipids by determination of the amount ofreaction product, phosphatidic acid. Alternatively, monitoring of thereaction could be performed by using fluorescently labeled substrate oracceptor molecules, which can be identical to or different fromArachidonoyl CoA and lysophosphatidic acid. Determination of LPAATactivity can either be performed in cell-based assays using endophilinoverexpressing cells or in in vitro assays using purified endophilin,generated as described above. Another target mechanism would be theinteraction of endophilin with other cellular proteins, which could befor example, but not exclusively, the β1 adrenergic receptor (Tang etal. (1999) PNAS 96:12559-12564) or CIN85 complexed with or without Cbl(Petrelli et al. (2002) Nature 416:187-190, Soubeyran et al (2002)

Nature 41 6:183-187). Methods for determining protein-proteinInteraction are well known In the art. For example binding of afluorescently labeled peptide derived from the protein to endophilin, orvice versa, could be detected by a change In polarisation. In case thatboth binding partners, which can be either the full length proteins aswell as one binding partner as the full length protein and the otherjust represented as a peptide are fluorescently labeled, bindung couldbe detected by fluorescence energy transfer (FRET) from one fluorophoreto the other. In addition, a variety of commercially available assayprinciples suitable for detection of protein-protein Interaction arewell known In the art, for example but not exclusively AlphaScreen(PerkinElmer) or Scintillation Proximity Assays (SPA) by Amersham.Alternatively, the Interaction of Endophilin with cellular proteinscould be the basis for a cell-based screening assay, In which bothproteins are fluorescently labeled and Interaction of both proteins Isdetected by-analysing cotranslocation of both proteins with a cellularImaging reader, as has been developed for example, but not exclusively,by Cellomics or EvotecOAI.

Of particular interest are screening assays for agents that have a lowtoxicity for mammalian cells. The term “agent” as used herein describesany molecule, e.g. protein or pharmaceutical, with the capability ofaltering or mimicking the physiological function of one or more of theproteins of the invention. Candidate agents encompass numerous chemicalclasses, though typically they are organic molecules, preferably smallorganic compounds having a molecular weight of more than 50 and lessthan about 2,500 Daltons. Candidate agents comprise functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise carbocyclic orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups.

Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acidsand derivatives, structural analogs or combinations thereof. Candidateagents are obtained from a wide variety of sources including librariesof synthetic or natural compounds. For example, numerous means areavailable for random and directed synthesis of a wide variety of organiccompounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Where the screening assay is a binding assay, one ormore of the molecules may be joined to a label, where the label candirectly or indirectly provide a detectable signal.

Another technique for drug screening, which may be used, provides forhigh throughput screening of compounds having suitable binding affinityto the protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to the proteins of the inventionlarge numbers of different small test compounds, e.g. aptamers,peptides, low-molecular weight compounds etc., are provided orsynthesized on a solid substrate, such as plastic pins or some othersurface. The test compounds are reacted with the proteins or fragmentsthereof, and washed. Bound proteins are then detected by methods wellknown in the art. Purified proteins can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support. In another embodiment, onemay use competitive drug screening assays in which neutralizingantibodies capable of binding the protein specifically compete with atest compound for binding the protein. In this manner, the antibodiescan be used to detect the presence of any peptide, which shares one ormore antigenic determinants with the protein.

The nucleic acids encoding the proteins of the invention can be used togenerate transgenic cell lines and animals. These transgenic animals areuseful in the study of the function and regulation of the proteins ofthe invention in vivo. Transgenic animals, particularly mammaliantransgenic animals, can serve as a model system for the investigation ofmany developmental and cellular processes common to humans. A variety ofnon-human models of metabolic disorders can be used to test modulatorsof the protein of the invention. Misexpression (for example,overexpression or lack of expression) of the protein of the invention,particular feeding conditions, and/or administration of biologicallyactive compounts can create models of metablic disorders.

In one embodiment of the invention, such assays use mouse models ofinsulin resistance and/or diabetes, such as mice carrying gene knockoutsin the leptin pathway (for example, ob (leptin) or db (leptin receptor)mice). Such mice develop typical symptoms of diabetes, show hepaticlipid accumulation and frequently have increased plasma lipid levels(see Bruning et al, 1998, Mol. Cell. 2:449-569). Susceptible wild typemice (for example C57BI/6) show similiar symptoms if fed a high fatdiet. In addition to testing the expression of the proteins of theinvention in such mouse strains (see EXAMPLE 4), these mice could beused to test whether administration of a candidate modulator alters forexample lipid accumulation in the liver, in plasma, or adipose tissuesusing standard assays well known in the art, such as FPLC, calorimetricassays, blood glucose level tests, insulin tolerance tests and others.

Transgenic animals may be made through homologous recombination inembryonic stem cells, where the normal locus of the gene encoding theprotein of the invention is mutated. Alternatively, a nucleic acidconstruct encoding the protein is injected into oocytes and is randomlyintegrated into the genome. One may also express the genes of theinvention or variants thereof in tissues where they are not normallyexpressed or at abnormal times of development. Furthermore, variants ofthe genes of the invention like specific constructs expressinganti-sense molecules or expression of dominant negative mutations, whichwill block or alter the expression of the proteins of the invention maybe randomly integrated into the genome. A detectable marker, such as lacZ or luciferase may be introduced into the locus of the genes of theinvention, where upregulation of expression of the genes of theinvention will result in an easily detectable change in phenotype.Vectors for stable integration include plasmids, retroviruses and otheranimal viruses, yeast artificial chromosomes (YACs), and the like. DNAconstructs for homologous recombination will contain at least portionsof the genes of the invention with the desired genetic modification, andwill include regions of homology to the target locus. Conveniently,markers for positive and negative selection are included. DNA constructsfor random integration do not need to contain regions of homology tomediate recombination. DNA constructs for random integration willconsist of the nucleic acids encoding the proteins of the invention, aregulatory element (promoter), an intron and a poly-adenylation signal.Methods for generating cells having targeted gene modifications throughhomologous recombination are known in the field. For embryonic stem (ES)cells, an ES cell line may be employed, or embryonic cells may beobtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Suchcells are grown on an appropriate fibroblast-feeder layer and are grownin the presence of leukemia inhibiting factor (LIF). ES or embryoniccells may be transfected and can then be used to produce transgenicanimals. After transfection, the ES cells are plated onto a feeder layerin an appropriate medium. Cells containing the construct may be selectedby employing a selection medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination. Colonies that are positive may then be used for embryomanipulation and morula aggregation. Briefly, morulae are obtained from4 to 6 week old superovulated females, the Zona Pellucida is removed andthe morulae are put into small depressions of a tissue culture dish. TheES cells are trypsinized, and the modified cells are placed into thedepression closely to the morulae. On the following day the aggregatesare transfered into the uterine horns of pseudopregnant females. Femalesare then allowed to go to term. Chimeric offsprings can be readilydetected by a change in coat color and are subsequently screened for thetransmission of the mutation into the next generation (F1-generation).Offspring of the F1-generation are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogenic or congenic grafts or transplants, or in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimal, domestic animals, etc., for example, mouse, rat, guinea pig,sheep, cow, pig, and others. The transgenic animals may be used infunctional studies, drug screening, and other applications and areuseful in the study of the function and regulation of the proteins ofthe invention in vivo.

Finally, the invention also relates to a kit comprising at least one of

-   -   (a) a Endophilin 3, Endophilin 1, or Endophilin 2 nucleic acid        molecule or a fragment thereof;    -   (b) a vector comprising the nucleic acid of (a);    -   (c) a host cell comprising the nucleic acid of (a) or the vector        of (b);    -   (d) a polypeptide encoded by the nucleic acid of (a);    -   (e) a fusion polypeptide encoded by the nucleic acid of (a);    -   (f) an antibody, an aptamer or another receptor against the        nucleic acid of (a) or the polypeptide of (d) or (e) and    -   (g) an anti-sense oligonucleotide of the nucleic acid of (a).

The kit may be used for diagnostic or therapeutic purposes or forscreening applications as described above. The kit may further containuser instructions.

THE FIGURES SHOW

FIG. 1 shows the increase of triglyceride content of EP(3)0593 fliescaused by heterozygous lethal integration of the P-vector (in comparisonto controls without integration of this vector).

FIG. 2 shows the molecular organisation of the mutated Endophilin genelocus.

FIG. 3. Endophilin Sequences

FIG. 3A. Nucleic Acid sequence of human Endophilin 3 (also referred toas SH3-domain GRB2-like 3 protein; GenBank Accession NumberNM_(—)003027; SEQ ID NO. 1).

FIG. 3B. Amino Acid sequence of human Endophilin 3 (also referred to asSH3-domain GRB2-like 3 protein; GenBank Accession Number NP_(—)003018;SEQ ID NO. 2).

FIG. 3C. Nucleic Acid sequence of human Endophilin 1 (also referred toas SH3-domain GRB2-like 1 protein; GenBank Accession NumberNM_(—)003025; SEQ ID NO. 3).

FIG. 3D. Amino Acid sequence of human Endophilin 1 (also referred to asSH3-domain GRB2-like 1 protein; GenBank Accession Number NP_(—)003016;SEQ ID NO. 4).

FIG. 3E. Nucleic Acid sequence of human Endophilin 2 (also referred toas SH3-domain GRB2-like 2 protein; GenBank Accession NumberNM_(—)003026; SEQ ID NO. 5).

FIG. 3F. Amino Acid sequence of human Endophilin 2 (also referred to asSH3-domain GRB2-like 2 protein; GenBank Accession Number NP_(—)003017;SEQ ID NO. 6).

FIG. 3G shows the CLUSTAL X (1.81) multiple sequence alignment forEndophilin. NP_(—)003016 refers to the Accession Number for humanEndophilin 1, NP_(—)003017 refers to the Accession Number for humanEndophilin 2, NP_(—)003018 refers to the Accession Number for humanEndophilin 3, and CG14296 refers to the GadFly Accession Number forDrosophila Endophilin A.

FIG. 4 shows the expression of Endophilin 3 in different mammalianmodels.

FIG. 4A. Expression of Endophilin 3 in different wildtype mouse tissues

FIG. 4B. Expression of Endophilin 3 in different mouse models: wildtype(wt), fasted mice and obese mice-(ob/ob)

FIG. 4C. Expression of Endophilin 3 in susceptible wild-type mice undera high-fat diet

FIG. 4D. Expression of Endophilin 3 during differentiation of cultured3T3-L1 cells from pre-adipocytes to mature adipocytes

FIG. 4E. Expression of Endophilin 3 during differentiation of cultured3T3-F422A cells from pre-adipocytes to mature adipocytes

FIG. 5 shows the expression of Endophilin 1 in different mammalianmodels.

FIG. 5A. Expression of Endophilin 1 in different wildtype mouse tissues

FIG. 5B. Expression of Endophilin 1 in different mouse models (inwildtype (wt) and obese (ob/ob) mice

FIG. 5C. Expression of Endophilin 1 in mice under a high-fat diet

FIG. 6. Expression of Endophilin 2 in different wildtype mouse tissues

FIG. 7 shows in vitro assays for the determination of triglyceride,glycogen and glucose levels in cells overexpressing endophilin 3.

FIG. 7A shows an increase in triglyceride levels in cells overexpressingendophilin 3 versus control cells. The Y-axis shows cellulartriglyceride levels (g/mg protein) and the X-axis shows days of celldifferentiation.

FIG. 7B shows an increase in glycogen levels in cells overexpressingendophilin 3. The Y-axis shows glycogen levels (shown as microg glycogenper mg protein) and the X-axis shows days of cell differentiation.

FIG. 7C shows an increase in glucose uptake in cells overexpressingendophilin 3. The Y-axis shows glucose levels (shown as disintegrationsper minutes -dpm- per mg protein) and the X-axis shows days of celldifferentiation.

THE EXAMPLES ILLUSTRATE THE INVENTION Example 1 Measurement ofTriglyceride Content in Drosophila (FIG. 1)

Mutant flies are obtained from the P Insertion Mutation Stock Center,Sezged, Hungary. The flies are grown under standard conditions known tothose skilled in the art. In the course of the experiment, additionalfeedings with bakers yeast (Saccharomyces cerevisiae) are provided. Theaverage increase of triglyceride content of Drosophila containing theEP(3)0593 vectors in heterozygous integration was investigated incomparison to control flies (FIG. 1). For determination of triglyceride,flies were incubated for 5 min at 90° C. in an aqueous buffer using awaterbath, followed by hot extraction. After another 5 min incubation at90° C. and mild centrifugation, the triglyceride content of the fliesextract was determined using Sigma Triglyceride (INT 336-10 or -20)assay by measuring changes in the optical density according to themanufacturer's protocol. As a reference protein content of the sameextract was measured using BIO-RAD DC Protein Assay according to themanufacturer's protocol. The assay was repeated several times. Theaverage triglyceride level of all flies of the EP collection (referredto as ‘EP-controls males’) is shown as 100% in FIG. 1. EP(3)0593heterozygous flies show constantly a higher triglyceride content thanthe controls (100%; column 2 in FIG. 1. Therefore, the loss of geneactivity in the locus 91D4 on chromosome 3R where the EP-vector ofEP(3)0593 flies is heterozygous lethal integrated, is responsible forchanges in the metabolism of the energy storage triglycerides, thereforerepresenting an model for obese flies.

Example 2 Identification of a Drosophila Gene Responsible for the Changein Triglyceride Content (FIG. 2)

In FIG. 2, genomic DNA sequence is represented by the assembly as adotted black line (from position 14653366 to 14678366 on chromosome 3R)that includes the integration sites of vector for lines EP(3)0593.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the lower two lines. Predicted exons of the cDNA with GadFlyAccession Number CG14296 are shown as dark grey bars and introns aslight grey bars. Endophilin A encodes for a gene that is predicted byGadFly sequence analysis programs as Accession Number CG14296. PublicDNA sequence databases (for example, NCBI GenBank or GadFly database)were screened thereby identifying the integration sites of linesEP(3)0593, causing an increase of triglyceride content. EP(3)0593 isintegrated 45 base pairs of in the 5′ exon of the cDNA with AccessionNumber CG 14296. Therefore, expression of the cDNA encoding AccessionNumber CG14296 could be effected by heterozygous integration of vectorsof lines EP(3)0593, leading to increase of the energy storagetriglycerides.

Example 3 Identification of Human Endophilin Homologues (FIG. 3)

Endophilin homologous proteins and nucleic acid molecules codingtherefore are obtainable from insect or vertebrate species, e.g. mammalsor birds. Particularly preferred are human Endophilin homologous nucleicacids, particularly nucleic acids encoding a human Endophilin 3 protein(Genbank Accession No. NM_(—)003027; Genbank Accession No. AF036269;EEN-B2-L1, SH3-domain GRB2-like 3), a human Endophilin 1 protein(Genbank Accession No. NM_(—)003025; Genbank Accession No. AF036268;EEN-B1, SH3-domain GRB2-like 1), or a human Endophilin 2 protein(Genbank Accession No. NM_(—)003026; Genbank Accession No. U65999; EEN,SH3-domain GRB2-like 2). Also preferred are mouse Endophilin homologousnucleic acids and polypeptides encoded thereby (Endophilin 3, GenBankAccession Number U58887; Endophilin 1, GenBank Accession Number U58886;or Endophilin 2, GenBank Accession Number U58885). Comparisons (ClustalX 1.8 analysis or Clustal W 1.82 analysis, see for example Thompson J.D. et al., (1 994) Nucleic Acids Res. 22(22):4673-4680; Thompson J. D.,(1997) Nucleic Acids Res 25(24):4876-4882; Higgins, D. G. et al., (1996)Methods Enzymol. 266:383-402) between the respective proteins ofdifferent species (human, mouse, and Drosophila) were conducted and analignment is shown in FIG. 3G.

Example 4 Expression of Endophilin Polypeptides in Mammalian (Mouse)Tissues (FIG. 4, FIG. 5, and FIG. 6)

For analyzing the expression of Endophilins in mammalian tissues,several mouse strains (preferably mice strains C57BI/6J, C57BI/6 ob/oband C57BI/KS db/db which are standard model systems in obesity anddiabetes research) were purchased from Harlan Winkelmann (33178 Borchen,Germany) and maintained under constant temperature (preferably 22° C.),40 per cent humidity and a light/dark cycle of preferably 14/10 hours.The mice were fed a standard chow (for example, from ssniffSpezialitäten GmbH, order number ssniff M-Z V1126-000). For the fastingexperiment (“fasted wild type mice”), wild type mice were starved for 48h without food, but only water supplied ad libitum. (see, for example,Schnetzler et al. J Clin Invest July 1993; 92(1):272-80, Mizuno et al.Proc Natl Acad Sci USA Apr. 1996 16;93(8):3434-8). Animals weresacrificed at an age of 6 to 8 weeks. The animal tissues were isolatedaccording to standard procedures known to those skilled in the art, snapfrozen in liquid nitrogen and stored at −80° C. until needed.

For analyzing the role of Endophilins in the in vitro differentiation ofdifferent mammalian cell culture cells for the conversion ofpre-adipocytes to adipocytes, mammalian fibroblast (3T3-L1) cells (e.g.,Green & Kehinde, Cell 1: 1 13-116, 1974) were obtained from the AmericanTissue Culture Collection (ATCC, Hanassas, Va., USA; ATCC-CL 173).3T3-L1 cells were maintained as fibroblasts and differentiated intoadipocytes as described in the prior art (e.g., Qiu. et al., J. Biol.Chem. 276:11988-95, 2001; Slieker et al., BBRC 251: 225-9, 1998). Inbrief, cells were plated in DMEM/10% FCS (Invitrogen, Karlsruhe,Germany) at 50,000 cells/well in duplicates in 6-well plastic dishes andcultured in a humidified atmosphere of 5% CO2 at 37° C. At confluence(defined as day 0: d0) cells were transferred to serum-free (SF) medium,containing DMEM/HamF12 (3:1; Invitrogen), Fetuin (300 μg/ml; Sigma,Munich, Germany), Transferrin (2 μg/ml; Sigma), Pantothenate (17 μM;Sigma), Biotin (1 μM; Sigma), and EGF (0.8 nM; Hoffmann-La Roche, Basel,Switzerland). Differentiation was induced by adding Dexamethasone (DEX;1 μM; Sigma), 3-Methyl-Isobutyl-1-Methylxanthine (MIX; 0.5 mM; Sigma),and bovine Insulin (5 μg/ml; Invitrogen). Four days after confluence(d4), cells were kept in SF medium, containing bovine Insulin (5 μg/ml)until differentiation was completed. At various time points of thedifferentiation procedure, beginning with day 0 (day of confluence) andday 2 (hormone addition; for example, dexamethason and3-isobutyl-1-methylxanthin), up to 10 days of differentiation, suitablealiquots of cells were taken every two days.

Alternatively, mammalian fibroblast 3T3-F442A cells (e.g., Green &Kehinde, Cell 7: 105-113, 1976) were obtained from the Harvard MedicalSchool, Department of Cell Biology (Boston, Mass., USA). 3T3-F442A cellswere maintained as fibroblasts and differentiated into adipocytes asdescribed previously (Djian, P. et al., J. Cell. Physiol., 124:554-556,1985). At various time points of the differentiation procedure,beginning with day 0 (day of confluence and hormone addition, forexample, Insulin), up to 1 0 days of differentiation, suitable aliquotsof cells were taken every two days. 3T3-F442A cells are differentiatingin vitro already in the confluent stage after hormone (insulin)addition.

For TaqMan Analysis of the proteins of the invention (FIG. 4, FIG. 5,and FIG. 6), RNA was isolated from mouse tissues or cell culture cellsusing Trizol Reagent (for example, from Invitrogen, Karlsruhe, Germany)and further purified with the RNeasy Kit (for example, from Qiagen,Germany) in combination with an DNase-treatment according to theinstructions of the manufacturers and as known to those skilled in theart. Total RNA was reverse transcribed (preferably using Superscript IIRNaseH-Reverse Transcriptase, from Invitrogen, Karlsruhe, Germany) andsubjected to Taqman analysis preferably using the Taqman 2×PCR MasterMix (from Applied Biosystems, Weiterstadt, Germany; the Mix containsaccording to the Manufacturer for example AmpliTaq Gold DNA Polymerase,AmpErase UNG, dNTPs with dUTP, passive reference Rox and optimizedbuffer components) on a GeneAmp 5700 Sequence Detection System (fromApplied Biosystems, Weiterstadt, Germany).

For the analysis of the expression of Endophilin 3, Endophilin 1, andEndophilin 2, taqman analysis was performed using the followingprimer/probe pairs: Mouse Endophilin 3 forward primer 5′-AAC GAG TAA ATTGCG CCC AT-3′; (Seq ID NO: 7) Mouse Endophilin 3 reverse primer 5′-CAAAGG GTG CGT TCC CAC T-3′; (Seq ID NO: 8) Mouse Endophilin 3 Taqman probe(5/6-FAM) CGA ATG GCC TGG GTA GTC (Seq ID NO: 9) CTT GAC TG (5/6-TAMRA)Mouse Endophilin 1 forward primer 5′-TGC TTT GGT AAT GCT GCT TCC-3′;(Seq ID NO: 10) Mouse Endophilin 1 reverse primer 5′-GTG GGC TTG GTG ACTCAT CC-3′; (Seq ID NO: 11) Mouse Endophilin 1 Taqman probe (5/6-FAM) ACATCA CGA ATG CAG GCC (Seq ID NO: 12) GCA G (5/6-TAMRA) Mouse Endophilin 2forward primer 5′-CGA CGA GAA CTG GTA TGA GGG-3′; (Seq ID NO: 13) MouseEndophilin 2 reverse primer 5′-GCA CGT AGC TGA GTG GGA AGA-3′; (Seq IDNO: 14) Mouse Endophilin 2 Taqman probe (5/6-FAM) ATG CTG CAC GGC CAATCA (Seq ID NO: 15) GGC (5/6-TAMRA)

Expression profiling studies showed that Endophilin 3 and Endophilin 1are clearly involved in the regulation of energy metabolism in mamals.In comparison to Endophilin 2, which is rather ubiquitously expressed,both Endophilin 3 and Endophilin 1 are more restricted in neuronaltissues. However, both proteins are also clearly expressed in WAT andBAT (FIG. 4A and FIG. 5A, respectively).

The expression of Endophilin 3 is strongly upregulated in liver offasted mice (see FIG. 4B). In addition, a marked downregulation can beobserved in brown adipose tissue of genetically obese (ob/ob) as well asof fasted mice (FIG. 4B). Endophilin 1 is strongly downregulated inwhite adipose tissue of genetically obese db/db mice (FIG. 5B).

The most prominent response with regard to metabolically active tissuescan be seen in mice hold under a high fat diet: For Endophilin 3 as wellas Endophilin 1, a dramatic decrease in their relative expression inwhite adipose tissue (WAT) can be observed in these mice (see FIG. 4Cand FIG. 5C, respectively).

With regard to changes in expression intensity during thedifferentiation of preadipocytes to adipocytes, a strong reduction inrelative signal intensity can be observed for Endophilin 3 during the invitro differentiation program of 3T3-L1 as well as 3T3-F442A cells (seeFIG. 4D and FIG. 4E).

Example 5 Assays for the Determination of Triglyceride Storage,Synthesis and Transport (FIG. 7A)

Retroviral Infection of Preadipocytes

Packaging cells were transfected with retroviral plasmids pLPCX carryingmouse Endophillin transgene and a selection marker using calciumphosphate procedure. Control cells were infected with pLPCX carrying notransgene. Briefly, exponentially growing packaging cells were seeded ata density of 350,000 cells per 6-well in 2 ml DMEM+10% FCS one daybefore transfection. 10 min before transfection chloroquine was addeddirectly to the overlying medium (25 μM end concentration). A 250 μltransfection mix consisting of 5 μg plasmid-DNA (candidate:helper-virusin a 1:1 ratio) and 250 mM CaCl₂ was prepared in a 15 ml plastic tube.The same volume of 2×HBS (280 μM NaCl, 50 μM HEPES, 1.5 mM Na₂HPO₄, pH7.06) was added and air bubbles were injected into the mixture for 15sec. The transfection mix was added drop wise to the packaging cells,distributed and the cells were incubated at 37° C., 5% CO₂ for 6 hours.The cells were washed with PBS and the medium was exchanged with 2 mlDMEM+10% CS per 6-well. One day after transfection the cells were washedagain and incubated for 2 days of virus collection in 1 ml DMEM+10% CSper 6-well at 32° C., 5% CO₂.

The supernatant was then filtered through a 0.45 μm cellulose acetatefilter and polybrene (end concentration 8 μg/ml) was added. Mammalianfibroblast (3T3-L1) cells in a sub-confluent state were overlaid withthe prepared virus containing medium. The infected cells were selectedfor 1 week with 2 μg/ml puromycin. Following selection the cells werechecked for transgene expression by western blot and immunofluorescence.Over expressing cells were seeded for differentiation.

3T3-L1 cells were maintained as fibroblasts and differentiated intoadipocytes as described in the prior art and in example 8. For analysingthe role of the proteins disclosed in this invention in the in vitroassays for the determination of triglyceride storage, synthesis andtransport were performed.

Preparation of Cell Lysates for Analysis of Metabolites

Starting at confluence (D0), cell media was changed every 48 hours.Cells and media were harvested 8 hours prior to media change as follows.Media was collected, and cells were washed twice in PBS prior to lysesin 600 microl HB-buffer (0.5% Polyoxyethylene 10 tridecylethane, 1 mMEDTA, 0.01M NaH2PO4, pH 7.4). After inactivation at 70° C. for 5minutes, cell lysates were prepared on Bio 101 systems lysing matrix B(0.1 mm silica beads; Q-Biogene, Carlsbad, USA) by agitation for 2×45seconds at a speed of 4.5 (Fastprep FP120, Bio 101 Thermosavant,Holbrock, USA). Supernatants of lysed cells were collected aftercentrifugation at 3000 rpm for 2 minutes, and stored in aliquots forlater analysis at −80° C.

Changes in cellular triglyceride levels during adipogenesis (FIG. 7A)Cell lysates and media were simultaneously analysed in 96-well platesfor total protein and triglyceride content using the Bio-Rad DC Proteinassay reagent (Bio-Rad, Munich, Germany) according to the manufacturer'sinstructions and a modified enzymatic triglyceride kit (GPO-Trinder;Sigma) briefly final volumes of reagents were adjusted to the 96-wellformat as follows: 10 μl sample was incubated with 200 μl reagent A for5 minutes at 37° C. After determination of glycerol (initial absorbanceat 540 nm), 50 μl reagent B was added followed by another incubation for5 minutes at 37° C. (final absorbance at 540 nm). Glycerol andtriglyceride concentrations were calculated using a glycerol standardset (Sigma) for the standard curve included in each-assay.

As shown in FIG. 7A, we found that in Endophilin 3 over-expressing cellss cellular triglyceride levels were increased throughout adipogenesis.

Example 6 Changes in Cellular Glycogen Levels During Adipogenesis (FIG.7B)

Cell lysates and media were simultaneously analysed in triplicates in96-well plates for total protein and glycogen content using the Bio-RadDC Protein assay reagent (Bio-Rad, Munich, Germany) according to themanufacturer's instructions and an enzymatic starch kit from Hoffmann-LaRoche (Basel, Switzerland). 10-μl samples were incubated with 20-μlamyloglucosidase solution for 15 minutes at 60° C. to digest glycogen toglucose. The glucose is further metabolised with 100μl distilled waterand 100 μl of enzyme cofactor buffer and 12 μl of enzyme buffer(hexokinase and glucose phosphate dehydrogenase). Background glucoselevels are determined by subtracting values from a duplicate platewithout the amyloglucosidase. Final absorbance is determined at 340 nm.HB-buffer as blank, and a standard curve of glycogen (Hoffmann-La Roche)were included in each assay. Glycogen content in samples was calculatedusing a standard curve.

As shown in FIG. 7B, we found that in Endophilin 3 over-expressing cellscellular glycogen levels were increased throughout adipogenesis.Glycogen levels in cells are more variable than triglyceride levelsbecause the turnover of glycogen is higher. Glucose is taken up by thecells rapidly and stored in the form of glycogen. This energy storage isthan primarily used for the metabolic demands of the cell. The increasein glycogen levels when over-expressing Endophilin 3 could be the effectof a decreased metabolic rate in these cells or an increase in glucoseuptake as compared to empty vector transduced cells.

Example 7 Glucose Uptake Assay (FIG. 7C)

For the determination of glucose uptake, cells were washed 3 times withPBS prior to serum starvation in Krebs-Ringer-Bicarbonate-Hepes buffer(KRBH; 134 nM NaCl, 3.5 mM KCl, 1.2 mM KH₂PO₄, 0.5 mM MgSO₄, 1.5 mMCaCl₂, 5 mM NaHCO₃, 10 mM Hepes, pH 7.4), supplemented with 0.1% FCS and0.5 mM Glucose for 2.5 h at 37° C. For insulin-stimulated glucoseuptake, cells were incubated with 1 μM bovine insulin (Sigma; carrier:0.005 N HCl) for 45 min at 37° C. Basal glucose uptake was determinedwith carrier only. Non-metabolizable 2-Deoxy-3H-D-Glucose (NEN LifeScience, Boston, USA) in a final activity of 0.4 μCi/Well/ml was addedfor 30 min at 37° C. For the calculation of background radioactivity, 25μM Cytochalasin B (Sigma) was used. All assays were performed induplicate wells. To terminate the reaction, cells were washed 3 timeswith ice cold PBS, and lysed in 1 ml 0.1N NaOH. Protein concentration ofeach well was assessed using the standard Biuret method (Protein assayreagent; Bio-Rad), and scintillation counting of cell lysates in 10volumes Ultima-gold cocktail (Packard Bioscience, Groningen,Netherlands) was performed.

As shown in FIG. 7C the basal glucose uptake of adipocyte (for example,3T3-L1) cells over-expressing Endophilin 3 is increased significantly bymore than 100% during adipogenesis at all times. This effect is alreadyvisible in the pre-adipocyte (d4) and also in a fully differentiatedadipocyte (d13). This increase in glucose and therefore energy uptake ofthe cells is most likely the reason for the increased glycogen andtriglyceride levels during adipocyte (e.g., 3T3-L1) differentiation (seeFIGS. 7 a and 7 b). Endophilin 3 does not seem to influence insulinstimulated glucose uptake but clearly has an effect on the glucoseuptake of adipocytes, confirming its role in diabetes and relatedmetabolic disorders.

Example 8 Generation and Analysis of Endophilin 3 Transgenic Animals(aP2-Endophilin 3)

Generation of the Transgenic Animals

Mouse Endophilin 3 cDNA was isolated from mouse midbrain tissue usingstandard protocols as known to those skilled in the art. The cDNA wasamplified by RT-PCR using the following primer pair: mEndophilin 3forward primer: 5′ GGC GCC GCC GCG ATG TCG GTG 3′ (SEQ ID NO: 16)mEndophilin 3 reverse primer: 5′ TAG GTC CAA AAG ACA CAT TTA CGG (SEQ IDNO: 17) AGG 3′.

The resulting mEndophilin 3 cDNA was cloned into the Smal site ofpTG-aP2-X-hgh-bgh-polyA (Develogen AG), a derivative of pBluescript KS+(Stratagene) containing additional restriction sites in the polylinker,an aP2-promoter/enhancer fragment (Graves et al., 1991), an exon/intronstructure derived from the human growth hormone gene and a bovinepolyadenylation signal according to standard protocols, resulting in aplasmid referred to as pTG-aP2-mEndophilinlll-hgh-bgh-polyA′.

The aP2-Endophilin 3 transgene was microinjected into the malepronucleus of fertilized mouse embryos (preferable strain FVB (HarlanWinkelmann)). Injected embryos were transferred into pseudo-pregnantfoster mice. Transgenic founders were detected by PCR analysis using anaP2 forward primer (SEQ ID NO: 18): 5′ TGC CAG GGA GAA CCA AAG T 3′ andEndophilin 3 reverse primer (SEQ ID NO: 19): 5′ GCC TCA CCA ACA TCT ACCAAC 3′. Transgenic mouse lines containing the aP2-Endophilin 3 constructwere established. Briefly, founder animals were backcrossed with C57/BL6mice to generate F1 mice for analysis. Transgenic mice were continouslybred onto the C57/B16 background.

Analysis of the Bodyweight of the Transgenic Mice

After weaning, male aP2-Endophilin transgenic mice and their wild-type(wt) littermates controls were placed in groups of 4 to 5 animals (N=4up to N=5) on control diet (preferably Altromin C1057 mod control! 4.5%crude fat) or high fat diet (preferably Altromin C1057 mod. high fat,23.5% crude fat). Total body weight of the animals was measured weeklyover a period of 12-16 weeks.

1. A pharmaceutical composition comprising a nucleic acid molecule ofthe Endophilin gene family or a polypeptide encoded thereby or afragment or a variant of said nucleic acid molecule or said polypeptideor an antibody, an aptamer or another receptor recognizing a nucleicacid molecule of the Endophilin gene family or a polypeptide encodedthereby together with pharmaceutically acceptable carriers, diluentsand/or adjuvants.
 2. The composition of claim 1, wherein the nucleicacid molecule is a vertebrate or insect Endophilin nucleic acid,particularly a human Endophilin homologous nucleic acids, particularlynucleic acids encoding a human Endophilin 3 protein (Genbank AccessionNo. NM_(—)003027; Genbank Accession No. AF036269), a human Endophilin 1protein (Genbank Accession No. NM_(—)003025; Genbank Accession No.AF036268) or a human Endophilin 2 protein (Genbank Accession No.NM_(—)003026; Genbank Accession No. U65999) and mouse Endophilinhomologous nucleic acids and polypeptides encoded thereby (Endophilin 3,GenBank Accession Number U58885; Endophilin 1, GenBank Accession NumberU58887; or Endophilin 2, GenBank Accession Number U58885;), or afragment thereof or a variant thereof.
 3. The composition of claim 1wherein said nucleic acid molecule (a) hybridizes at 50° C. in asolution containing 1×SSC and 0.1% SDS to a nucleic acid moleculeencoding as specified in claim 2 or a nucleic acid molecule which iscomplementary thereto; (b) it is degenerate with respect to the nucleicacid molecule of (a); (c) encodes a polypeptide which is at least 85%,preferably at least 90%, more preferably at least 95%, more preferablyat least 98% and up to 99.6% identical to an Endophilin as defined inclaim 2; (d) differs from the nucleic acid molecule of (a) to (c) bymutation and wherein said mutation causes an alteration, deletion,duplication or premature stop in the encoded polypeptide.
 4. Thecomposition of claim 1, wherein the nucleic acid molecule is a DNAmolecule, particularly a cDNA or a genomic DNA.
 5. The composition ofclaim 1, wherein said nucleic acid encodes a polypeptide contributing toregulating the energy homeostasis and/or the metabolism oftriglycerides.
 6. The composition of claim 1, wherein said nucleic acidmolecule is a recombinant nucleic acid molecule.
 7. The composition ofclaim 1, wherein the nucleic acid molecule is a vector, particularly anexpression vector.
 8. The composition of claim 1, wherein thepolypeptide is a recombinant polypeptide.
 9. The composition of claim 8,wherein said recombinant polypeptide is a fusion polypeptide.
 10. Thecomposition of claim 1, wherein said nucleic acid molecule is selectedfrom hybridization probes, primers and anti-sense oligonucleotides. 11.The composition of claim 1 which is a diagnostic composition.
 12. Thecomposition of claim 1 which is a therapeutic composition.
 13. Thecomposition of claim 1 for the manufacture of an agent for detectingand/or verifying, for the treatment, alleviation and/or prevention of andisorders, including metabolic diseases such as obesity and otherbody-weight regulation disorders as well as related disorders such aseating disorder, cachexia, diabetes mellitus, hypertension, coronaryheart disease, hypercholesterolemia, dyslipidemia, osteoarthritis,gallstones, cancer, e.g. cancers of the reproductive organs, and sleepapnea and others, in cells, cell masses, organs and/or subjects.
 14. Thecomposition of claim 13 for the manufacture of an agent for modulatingmetabolic processes, particularly metabolite transport in adipocytes oradipogenesis.
 15. Use of a nucleic acid molecule of the Endophilin genefamily or a polypeptide encoded thereby or a fragment or a variant ofsaid nucleic acid molecule or said polypeptide or an antibody, anaptamer or another receptor recognizing a nucleic acid molecule of theEndophilin gene family or a polypeptide encoded thereby for controllingthe function of a gene and/or a gene product which is influenced and/ormodified by an Endophilin homologous polypeptide.
 16. Use of the nucleicacid molecule of the Endophilin gene family or a polypeptide encodedthereby or a fragment or a variant of said nucleic acid molecule or saidpolypeptide or an antibody, an aptamer or another receptor recognizing anucleic acid molecule of the Endophilin gene family or a polypeptideencoded thereby for identifying substances capable of interacting withan Endophilin homologous polypeptide.
 17. A non-human transgenic animalexhibiting a modified expression of an Endophilin polypeptide.
 18. Theanimal of claim 17, wherein the expression of the Endophilin polypeptideis increased and/or reduced.
 19. A recombinant host cell exhibiting amodified expression of an Endophilin polypeptide.
 20. The cell of claim19 which is a human cell.
 21. A method of identifying a (poly)peptideinvolved in the regulation of energy homeostasis and/or metabolism oftriglycerides in a mammal comprising the steps of (a) contacting acollection of (poly)peptides with an Endophilin polypeptide or afragment thereof under conditions that allow binding of said(poly)peptides; (b) removing (poly)peptides which do not bind and (c)identifying (poly)peptides that bind to said Endophilin polypeptide orfragment thereof.
 22. The method of claim 21, wherein the (poly)peptidesbind to said Endophilin polypeptide or fragment thereof via an SH3domain-mediated interaction.
 23. A method of screening for an agentwhich modulates the interaction of an Endophilin polypeptide with abinding target/agent, comprising the steps of (a) incubating a mixturecomprising (aa) an Endophilin polypeptide, or a fragment thereof; (ab) abinding target/agent of said Endophilin homologous polypeptide orfragment thereof; and (ac) a candidate agent under conditions wherebysaid Endophilin polypeptide or fragment thereof specifically binds tosaid binding target/agent at a reference affinity; (b) detecting thebinding affinity of said Endophilin polypeptide or fragment thereof tosaid binding target to determine a (candidate) agent-biased affinity;and (c) determining a difference between (candidate) agent-biasedaffinity and reference affinity.
 24. The method of claim 23 wherein thebinding target/agent binds to the Endophilin polypeptide or fragmentthereof via an SH3 domain-mediated interaction.
 25. The method of claim23 wherein the agent is a small molecular modulator, a nucleic acidmodulator or an antibody.
 26. A method of screening for an agent whichmodulates the activity of an Endophilin polypeptide, comprising thesteps of (a) incubating a mixture comprising (aa) an Endophilinpolypeptide, or a fragment thereof and (ab) a candidate agent underconditions whereby said Endophilin polypeptide or fragment thereofexhibits a reference activity; (b) detecting the activity of saidEndophilin polypeptide or a fragment thereof to determine a (candidate)agent-biased activity and (c) determining a difference between(candidate) agent-biased activity and reference activity.
 27. The methodof claim 26, wherein the agent is a small molecular modulator, a nucleicacid modulator or an antibody.
 28. (canceled)
 29. (canceled)
 30. Use ofa (poly)peptide as identified by the method of claim 21 for thepreparation of a pharmaceutical composition for the treatment,alleviation and/or prevention of of diseases and disorders, includingmetabolic diseases such as obesity and other body-weight regulationdisorders as well as related disorders such as eating disorder,cachexia, diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer,e.g. cancers of the reproductive organs, and sleep apnea and otherdiseases and disorders.
 31. Use of a nucleic acid molecule of theEndophilin gene family or of a fragment thereof for the preparation of anon-human animal which over- or under-expresses the Endophilin geneproduct.
 32. Kit comprising at least one of (a) an Endophilin nucleicacid molecule or a fragment thereof; (b) a vector comprising the nucleicacid of (a); (c) a host cell comprising the nucleic acid of (a) or thevector of (b); (d) a polypeptide encoded by the nucleic acid of (a); (e)a fusion polypeptide encoded by the nucleic acid of (a); (f) anantibody, an aptamer or another receptor against the nucleic acid of (a)or the polypeptide of (d) or (e) and (g) an anti-sense oligonucleotideof the nucleic acid of (a).
 33. Use of an agent as identified by themethod of claim 23 for the preparation of a pharmaceutical compositionfor the treatment, alleviation and/or prevention of of diseases anddisorders, including metabolic diseases such as obesity and otherbody-weight regulation disorders as well as related disorders such aseating disorder, cachexia, diabetes mellitus, hypertension, coronaryheart disease, hypercholesterolemia, dyslipidemia, osteoarthritis,gallstones, cancer, e.g. cancers of the reproductive organs, and sleepapnea and other diseases and disorders.